We propose to use an innovative multiplex fluorescent in situ method for determining transcript numbers from multiple genes in the same cells, seqFISH, in a model system to address the general problem of how transcription factor effects are exerted on different classes of target genes in a dynamic developmental system. The proposal is a Revision for the existing, funded project, Genomic site binding rules and regulatory factor function in developing T cells (R01HD076915), and it adds a crucial single-cell analysis component to clarify the interpretation and extend the insights from the project. In parallel, the biological system offers many advantages for enhancing the power and demonstrating the utility of the seqFISH technique. We exploit a well-characterized framework of developmental events through which multipotent hematopoietic progenitors undergo commitment to become T-cell precursors, a system that can be studied in parallel in vivo and in vitro and which is highly defined at the cellular level and in terms of patterns of gene expression The project focuses on the crucial but enigmatic role of the transcription factor PU.1, which is needed to support the early stages in T-cell development but does so apparently at the price of maintaining a regulatory bridge to an alternative set of developmental fates, i.e. macrophage, granulocyte, and dendritic cell fates. PU.1 has lineage-specific differences in its patterns of genomic occupancy in different hematopoietic precursors, but many of its binding sites do not appear to be linked with function. The parent proposal combines acute perturbation assays, genome-wide RNA-seq, and diagnostic ChIP-seq approaches to determine the rules that relate PU.1 occupancy to PU.1 regulatory functions in this early T-cell context. However, it is important to determine how homogeneous each baseline state is, and how uniform PU.1 actions are on all the cells at a given early T-cell stage, to resolve which PU.1 target genes are actually responding to PU.1 in the same regulatory-state context and which are responding in a different one. To answer this question and reveal in detail how different target genes process changes in activity of the same regulator, we propose a new collaboration between the Ellen Rothenberg and Long Cai groups. Specific aim 1: Use seqFISH to characterize the variation in PU.1 expression in T-cell precursors and variation in levels of known and suspected PU.1 target genes, relative to expression of genes that can modulate PU.1 effects. Specific aim 2: Optimize seqFISH technology for detection of transcripts from >20 different genes per cell in individual cells. Specific aim 3: Analyze the effects of PU.1 deletion and PU.1 antagonism on expression of different classes of candidate target genes in single cells, and determine their correlation wit levels of different modulating factors in those cells.